The goal of the experiments described in this proposal is to elucidate the mechanisms by which tumor necrosis factor (TNF) induces cell death. We plan to focus on the events that occur within one to two hours of binding of TNF to the cell membrane. Although TNF-induced cell death is a lengthy process taking up to 24 hr to reach completion, it is likely that early events occurring within the cell set in motion the sequence of steps leading to cell death. In Specific Aim I, we will continue our exploration of the intracellular messengers that stimulate the cytolysis of C3HA cells, a cell line that undergoes a necrotic form of cell death. We will compare these events to those occurring in the TNF-mediated cell death of L929 cells, a cell line that undergoes apoptosis. Necrosis and apoptosis (or programmed cell death) are predominately distinguished by the intracellular cytolytic targets. In necrosis, extensive cytoplasmic and cytoskeletal disruption occurs during the cytolytic process; whereas, the nucleus is the major target in apoptosis. The experiments described in Specific Aim I are aimed at understanding the differences between these two common forms of cell death as they relate to TNF. In Specific Aim II, we continue our exploration of the mechanism(s) of resistance to TNF that are at work in most normal cells. We are particularly interested in comparing the signal transduction pathways that mediate TNF-induced necrosis to those that are important in growth factor-induced proliferation. We will ask whether proliferative mechanisms also work to protect cells from the cytolytic effects of TNF. Finally, as part of our work, we have identified a population of C3HA cells in which we observe spontaneous oscillations in [Ca2+]. We have discovered that these oscillations are inhibited by TNF in sensitized cells. In Specific Aim III, we will determine the relationship between this oscillatory behavior and TNF-induced necrosis. We will also use the spontaneously oscillating cells as model system for understanding [Ca2+] oscillations in general. Elucidation of the mechanisms of TNF- induced cell death will greatly increase our knowledge of the pathways by which the organism is able to identify and kill abnormal cells and at the same time protect itself from its own cytopathic molecules.